1. Chapter 5: DNA Replication, Repair, and Recombination

2. Maintenance of DNA Sequences
Long Term Survival of Species Vs Survival of the Individual

3. Maintenance of DNA Sequences
Methods for Estimating Mutation Rates
Rapid generation of bacteria makes possible to detect bact w/ specific gene mutation
Mutation in gene required for lactose metabolism detected using indicator dyes
Indirect estimates of mutation rate: comparisons of aa sequence of same protein across species
Better estimates:
1. comparisions aa sequences in protein whose aa sequence does not matter
2. comparisions DNA sequences in regions of genome that does not carry critical info

4. Maintenance of DNA Sequences
Many Mutations Are Deleterious & Eliminated
Ea protein exhibits own characteristic rate of evol which reflects probability that aa chg will be harmful
6-7 chgs harmful to cytochrome C
Every aa chg harmful to histones

5. Maintenance of DNA Sequences
Mutation Rates are Extremely Low
Mutation rate in bact and mammals = 1 nucleotide chg/109 nucleotides ea time DNA replicated
Low mutation rates essential for life
Many mutations deleterious, cannot afford to accumulate in germ cells
Mutation frequency limits number of essential proteins organism can encode ~60,000
Germ cell stability vs Somatic Cell Stability

6. Maintenance of DNA Sequences
Multicellular Organisms Dependent upon Hi Fidelity Maintenance Afforded By:
Accuracy of DNA replication and distribution
Efficiency of DNA repair enzymes

7. Maintenance of DNA Sequences
High Fidelity DNA Replication
Error rate= 1 mistake/109 nucleotides
Afforded by complementary base pairing and proof-reading capability of DNA polymerase

8. Maintenance of DNA Sequences
DNA Polymerase as Self Correcting Enzyme
Correct nucleotide greater affinity than incorrect nucleotide
Conformation Chg after base pairing causes incorrect nucleotide to dissociate
Exonucleolytic proofreading of DNA polymerase
DNA molecules w/ mismatched 3’ OH end are not effective templates; polymerase cannot extend when 3’ OH is not base paired
DNA polymerase has separate catalytic site that removes unpaired residues at terminus

9. Mechanism of DNA Replication
General Features of DNA Replication
Semiconservative
Complementary Base Pairing
DNA Replication Fork is Assymetrical
Replication occurs in 5’ ’ Direction

10. DNA Replication Okazaki Fragments
DNA Primase uses rNTPs to synthesize short primers on lagging Strand
Primers ~10 nucleotides long and spaced ~ bp
DNA repair system removes RNA primer; replaces it w/DNA
DNA ligase joins fragments

11. DNA Replication DNA Helicase
Hydrolyze ATP when bound to ssDNA and opens up helix as it moves along DNA
Moves 1000 bp/sec
2 helicases: one on leading and one on lagging strand
SSB proteins aid helicase by destabilizing unwound ss conformation

12. DNA Replication
SSB proteins help DNA helicase destabilizing ssDNA

13. DNA Replication DNA Polymerase held to DNA by clamp regulatory protein
Clamp protein releases DNA poly when runs into dsDNA
Forms ring around DNA helix
Assembly of clamp around DNA requires ATP hydrolysis
Remains on leading strand for long time; only on lagging strand for short time when it reaches 5’ end of proceeding Okazaki fragments

14. DNA Replication Replication Machine (1 x 106 daltons)
DNA replication accomplished by multienzyme complex that moves rapidly along DNA by nucleoside hydrolysis
Subunits include:
(2) DNA Polymerases
helicase
SSB
Clamp Protein
Increases efficiency of replication

15. DNA Replication Okazaki Fragments
RNA that primed synthesis of 5’ end removed
Gap filled by DNA repair enzymes
Ligase links fragments together

16. DNA Replication Strand Directed Mismatch Repair System
Removes replication errors not recognized by replication machine
Detects distortion in DNA helix
Distinguishes newly replicated strand from parental strand by methylation of A residues in GATC in bact
Methylation occurs shortly after replication occurs
Reduces error rate 100X
3 Step Process
recognition of mismatch
excision of segment of DNA containing mismatch
resynthesis of excised fragment

17. DNA Replication
Strand Directed Mismatch Repair

18. DNA Replication Strand Directed Mismatch Repair in Humans
Newly synthesized strand is preferentially nicked and can be distinguish in this manner from parental strand
Defective copy of mismatch repair gene predisposed to cancer

19. DNA Replication DNA Topoisomerases
Reversible nuclease that covalently adds itself to DNA phosphate backbone to break phosphodiester bond
Phosphodiester bond reforms as protein leaves
Two Types
Topoisomerase I- produces single stranded break
Topoisomerase II- produces transient double stranded break

20. DNA Replication
Topoisomerase I

21. DNA Replication
Topoisomerase II

22. DNA Replication Eucaryotes vs Procaryotes
Enzymology, fundamental features, replication fork geometry, and use of multiprotein machinery conserved
More protein components in Euk replication machinery
Replication must proceed through nucleosomes
O. fragments in Euk ~200 bp as opposed to Pro
Replication fork moves 10X faster in Pro

23. DNA Replication Initiation and Completion of DNA Replication in Chromosomes
DNA Replication Begins at Origins of Replication
Positions at which DNA helix first opened
In simple cells ori defined DNA sequence bp
Sequence attracts initiator proteins
Typically rich in AT base pairs

24. DNA Replication Initiation and Completion of DNA Replication in Chromosomes
Bacteria
Single Ori
Initiation or replication highly regulated
Once initiated replication forks move at ~ bp/sec
Replicate 4.6 x 106 bp in ~40 minutes

25. DNA Replication Initiation and Completion of DNA Replication in Chromosomes
Eukaryotic Chromosomes Have Multiple Origins of Replication
Relication forks travel at ~50 bp/sec
Ea chromosome contains ~150 million base pairs
Replication origins activate in clusters or replication units of ori’s
Individual ori’s spaced at intervals of 30, ,000 bp

26. DNA Replication Initiation and Completion of DNA Replication in Chromosomes
Eukarotic DNA replication During S phase
Ea chromo replicates to produce 2 copies that remain
joined at centromeres until M phase
S phase lasts ~8 hours
Diff regions on same chromosomes replicate at distinct
times during S phase
Replication btwn 2 ori’s takes ~ 1 hr
BrdU experiments
Highly condensed chromatin replicates late while less
condensed regions replicate early
Housekeeping and cell specific genes

27. DNA Replication Initiation and Completion of DNA Replication in Chromosomes
Replication Origins Well Defined Sequences in Yeast
ARS autonomously replicating sequence
ARS spaced 30,000 bp apart
ARS deletions slow replication
ORC origin recognition complex
marks replication origin
binds Mcm (DNA helicase)
Cdc6 (helicase loading factor)

28. DNA Replication Initiation and Completion of DNA Replication in Chromosomes
Mammalian DNA Sequences that Specify Initiation of Replication
1000’s bp in length
Can function when placed in regions where chromo not too condensed
Human ORC required for replication initiation also bind Cdc6 and Mcm proteins
Binding sites for ORC proteins less specific

29. DNA Replication Initiation and Completion of DNA Replication in Chromosomes
New Nucleosomes Assembled Behind Replication Fork
lg amt of new histone protein required during replication
20 repeated gene sets (H1, H2A, H2B, H3, H4)
Histones syn in S phase ( transcription, degradation)
Histone proteins remarkably stable
Remodeling complexes destabilize DNA histone interface
during replication
CAFs (chromatin assembly factors) assist in addition of new nucleosome behind replication fork

30. DNA Replication Initiation and Completion of DNA Replication in Chromosomes
Telomerase Replicates Ends of Chromosomes
Telomere DNA sequences contain many tandem repeat sequences
Human telomere sequence GGGTTTA extends 10,000 nucleotides
Telomerase= special reverse transcriptase
Telomerase elongates repeat sequence recognizing tip of G-rich strand uses RNA template that is a component of enzyme itself
Protruding 3’ end loops back to hid terminus and protect it from degradative enzymes

31. DNA Repair
Despite 1000’s of alterations that occur in DNA ea day, few are retained as
mutations
Efficient reapir mechanisms
Impt of DNA repair highlighted by:
# of genes devoted to DNA repair
mutation rates as a function of inactivation or loss of DNA repair gene
Defects in DNA repair associated w/ several disease states

32. DNA Repair Types of DNA Damage: Base Loss and Base Modification
Chemical Modification
Photodamage thymine dimer
Depurination
Chemical Modification by O2 free radicals
Deamination

33. DNA Repair DNA Glycosylases
Cleave glycosyl bond that connects base to backbone sugar to remove base
> 6 Different types including those that remove:
deaminated C’s different types of alkylated or oxidize bases
deaminated A’s bases w/ open rings
bases w/ C=C

34. DNA Repair Base Excision Repair
DNA glycosylase recognizes damaged base
Removes base leaving deoxyribose sugar
AP endonuclease cuts phosphodiester bkbone
DNA polymerase replaces missing nucleotide
DNA ligase seals nick

35. DNA Repair Nucleotide Excision Repair Bulky Lesion Recognition
Demarcation and unwinding
Assembly of Repair enzymes
Dual Incision
Release of Damaged Nucleotide
Gap Filling DNA Synthesis

36. DNA Repair Chemistry of DNA Bases Facilitates Damage Detection
RNA thot to be original genetic material A, C, G, U
Why U replaced w/ T?
Deaminated C converted to U
DNA repair system unable to distinguish daminated C from U in RNA

37. DNA Repair Repairing Double Stranded Breaks in DNA
Nonhomologous end-joining repair
original DNA sequence is altered during repair (deletions or insertions)
Homologous end-joining repair
general recombination mechanism; info transferred from intact strand

38. DNA Repair DNA Damage Can Activate Expression of Whole Sets of Genes
Heat Shock Response
SOS Response

39. DNA Repair DNA Damage Delays Progression of Cell Cycle
DNA damage generates signals that block cell cycle progression
Blocks occur to extend the time for DNA Repair
ATM ataxia telangiectasia- defects in gene encoding ATM protein

40. Recombination DNA sequences occasionally rearranged
Rearrangments may alter gene structure as well as timing and level of expression
Promote variation

41. Recombination Two Classes 1. General or Homologous Recombination
2. Site-Specific Recombination

42. Recombination General or Homologous Recombination
Exchange btwn homologous DNA sequences
Essential repair mechanism
Essential for chromosomal segregation
Very Precise
Crossing over creates new combinations of DNA seq on ea chromo

43. Recombination Major Steps in General or Homologous Recombination
1. Synapsis
2. Branch Chain Migration
3. Isomerization of Holliday Junction
4. Resolution

44. Recombination
General or Homologous Recombination Guided by Base Pairing Interactions
Cross over of DNA from different chromosomes
ds helices break and two broken ends join opp. partners to reform intact ds helices
Exchange occurs only if there is extensive sequence homology
No nucleotides are altered at site of exchange; no loss or gain

45. Recombination DNA Synapsis catalyzed by RecA Protein
DNA strand from one helix has been exposed and its nucleotides made available for pairing w/ another molec= synapsis
Initiated by endonuclease cutting two strands of DNA and 5’ end chewed back to form ss 3’ end
SSB proteins hold strands apart
RecA allows ssDNA to pair w/ homologous region of DNA=synapsis

46. Recombination RecA Proteins also Facilitate Branch Chain Migration
Unpaired region of one of the ss displaces paired region of other ss moving the point
RecA catalyzes unidirectional branch migration producing region of heteroduplex DNA 1000’s bp in length

47. Recombination Holliday Junction
Two homolgous DNA helices paired and held together by reciprocal exchg of two of the four strands
Two pairs of strands: one pair of crossing strands and one pair or noncrossing
Isomerization leads to open structure where both pairs occupy equivalent positions
Holliday junction resolved by cutting of helices

48. Recombination
Resolution of Holliday Junction

49. Recombination Site-Specific Recombination
Mobile genetic elements move btwn nonhomologous sequences
Molibe genetic elements
size range 100s-1000s bp
found in nearly all cells
some represent viral sequences
relics constitute significant portion of genome (repeat sequences)

50. Recombination Movement of Mobile Genetic Elements
Site specific recombo mediated by enzymes recognize short specific nucleotide sequences present in one or both of recombo DNA molec
No sequence homology required
Mobile genetic elements generally encode enzyme that guides movement and special sites upon which enzyme acts
Elements move by transposition or conservative mechanisms

51. Recombination
Transpositional vs Conservative Site Specific Recombination
Transpositional= breakage rxns at ends of mobile DNA segments and attachment of those ends at one of many diff nonhomologous target sites
Conservative= production of short heteroduplex joint and thus requires short DNA sequence that is the same on both donor and recipient DNA

52. Recombination Transpositional Site Specific Recombination
Can insert mobile genetic elements into any DNA sequence
transposase acts on specific DNA seq at ea end of transposon disconnecting it from flanking DNA and inserting into new location
Transposons move only rarely (once every 105 generations in bact)
3 Types of Transposons

53. Recombination DNA Only Transposons
Move by DNA breakage and joining “cut and paste” mechanism
Inverted repeat recognized at ends and brought together forming loop
Insertion catalyzed by transposase occurs at random sites through staggered breaks
Break resealed but breakage and repair often alters DNA sequence resulting in mutations at site of excision

54. Recombination Retroviral-like Retrotransposons
Resemble retroviruses but lack protein coat
Transcription of transposon into RNA
Transcript translated by host encodes RT that produces ds DNA
Linear ds DNA integrates into site on chromo using integrase also encoded by transposon

55. Recombination Nonretroviral Retrotransposons
L1 or LINE for long interspersed nuclear element
L1 RNA synthesis
Endonuclease attached to L1 RT and L1 RNA
Endonuclease nicks target DNA at insertion site
Released 3’ OH end used as primer for RT that generates ssDNA copy of element linked to target
Leads to synthesis of second DNA strand that is inserted where original nick was made

56. Recombination
Different Transponable Elements Predominate in Different Organisms
Bacterial transposons are of DNA only type w/ a few nonretroviral transposons
Yeast main mobile elements are retroviral retrotransposons
Drosophilia and humans contain all three types of tranposable elements